Background: The Repeat Expansion Diseases are caused by intergenerational expansionsof a specific tandem repeat. More than 20 such diseases that belong to this group havebeen identified thus far. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. Carriers of alleles with 55-200repeats, so-called premutation (PM) alleles, are at risk for a neurodegenerative disorder,Fragile X-associated tremor/ataxia syndrome (FXTAS) and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency (FXPOI). Furthermore, in females,the PM allele can undergo expansion on intergenerational transfer that can result in their children having alleles with much larger numbers of repeats. Such full mutation (FM) alleles become epigenetically silenced via a process we are still trying to understand. Silencing results in a deficiency of the protein product of this gene, FMRP,which is involved in, amongst other things, insulin signaling and glucose metabolism. The FMRP deficiency results in Fragile X syndrome, the most common heritable cause of intellectual disability and autism. The threshold for methylation is currently considered to be 200 repeats based on Southern blotting of DNA from individuals symptomatic for FXS. Progress report: In this reporting period we have carried out additional studies on the silencing mechanism. We have identified a number of new inhibitors of silencing and are currently following up on these compounds to pinpoint their mode of action. In the course of doing this work we realized that better tools were needed to properly characterize the cells we were working with and thus to properly address some of our scientific questions. We thus developed a number of new assays for determining the repeat size, methylation status and AGG-interruption profile that we think will be useful both for basic research and in clinic. In addition to our work on gene silencing, we have also extended our previous work on the pathology seen in a mouse model of the FXDs to show that these animals have mitochondrial defects that could contribute to both FXTAS and FXPOI. This is significant since these mice do not make the toxic Repeat-associated Non-AUG (RAN) protein that is made in human PM carriers and in other mouse models. Thus, our data support the idea that the FMR1 transcript may indeed have intrinsically deleterious effects independent of the ability to make this abnormal protein. We have also identified factors that affect the hyper-expression of the PM allele. Future work will focus on developing a deeper understanding of the relevance of these factors for the etiology of FXTAS and FXPOI.